Insulin Signalling and Regulation of Protein Kinase B in Adipocytes

Insulin resistance is a hallmark of type 2 diabetes, an increasingly common disorder. The cause of insulin resistance is supposedly failures in the processes used by insulin to signal to the interior of its target cells. These failing steps are still unknown, most probably because of incomplete knowledge of how the insulin signals are transmitted. Since insulin resistance is strongly linked to obesity, defects in lipid metabolism or other adipocyte functions, may be an important factor in the development of this pathological states. It is therefore of particular interest to study insulin signalling and lipid metabolism in adipose tissue. More specifically, the aim of this thesis was to study the regulation of adipocyte protein kinase B (PKB), an insulin-stimulated kinase that has been implicated in mediating many of insulin's metabolic as well as mitogenic effects. We have shown, that in response to insulin, adipocyte PKB translocates from the cytosol to the plasma membrane in a phosphoinositide 3-kinase (PI3-K)-dependent manner. This is believed to induce a conformational change in PKB, allowing it to be phosphorylated and activated by the upstream kinases phosphoinositide-dependent kinase (PDK) -1 and 2. We have demonstrated that PKBß in primary adipocytes is unphosphorylated prior to stimulation, and insulin mainly induces phosphorylation on Ser-474. Furthermore, protein phosphatase 2A (PP2A) was identified as the phosphatase responsible for dephosphorylation and deactivation of PKB in adipocytes. In addition, we have initiated an investigation regarding the regulation and role of PDK1 in adipocytes. Endogenous PDK1 was shown not to be activated, but to translocate from the cytosol to the membrane fraction, in response to insulin. Moreover, adenoviral-mediated expression of PDK1 was used in order to assess the role of PDK1 in primary adipocytes. A recent study has been focused on the kinase inhibitor dimethylaminopurine (DMAP), and its effects on metabolic signalling pathways in adipocytes. DMAP was demonstrated to inhibit insulin-induced glucose uptake, antilipolysis and lipogenesis. Possible molecular targets, inhibition of which may mediate the effects of DMAP, were shown to be PKB and c-jun N-terminal kinase (JNK). In summary, this thesis has provided valuable information regarding the molecular mechanisms underlying insulin-induced activation of PKB, a key component of the insulin signalling pathway.